Antiviral combinations

ABSTRACT

Combinations of (2R, cis)-4-amino-1-(2-hydmxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one or a pharmaceutically acceptable derivative thereof and 11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,3-b;2 1 ,3 1  -e][1,4]diazepin-6-one or a pharmaceutically acceptable derivative thereof, pharmaceutical formulations of such combinations and the use of such combinations and formulations to treat viral infections, in particular HIV infections, are described.

This application is a 371 of PCT/EP93/01897 filed on Jul. 13, 1993.

The present invention relates to combinations of antiviral agents. Morespecifically it is concerned with combinations of 1,3-oxathiolanenucleoside analogues with other antiviral agents, in particular agentseffective against HIV. Human immunodefidency virus (HIV) causes avariety of clinical conditions including the acquired immune deficiencysyndrome (AIDS) and chronic neurological disorders. Nucleosides such asAZT, ddC and ddl inhibit HIV replication in vitro, and appear to exerttheir antiviral activity on the virus-encoded reverse transcriptaseenzyme after metabolism by the cell to their 5'-triphosphatederivatives.

AZT reduces morbidity and mortality in patients with AIDS. However, HIVinfection of cells results in integration of the virus genome into thehost chromosome, and so it has been necessary to continue AZT treatmentfor long periods of time. The consequences of long-term AZT therapy areassociated bone-marrow toxicity and the appearance of AZT-resistantvariants of HIV-1. Similarly, some AIDS patients treated with ddCdevelop peripheral neuropathy and ddl has been shown to inducepancreatitis and peripheral neuropathy.

The use of combinations of compounds may give rise to an equivalentantiviral effect with reduced toxicity, or an increase in drug efficacyif synergy between compounds occurs. Lower overall drug doses willpossibly also reduce the frequency of occurrence of drug-resistantvariants of HIV. Many different methods have been used to examine theeffects of combinations of compounds acting together in different assaysystems. All of these methods have limitations and for example, somemethods have been applied to systems other than those for which theywere derived. AZT demonstrates synergistic antiviral activity in vitroin combination with agents that act at HIV-1 replicative steps otherthan reverse transcription, including recombinant soluble CD4castanospermine and recombinant interferon alpha. However, it must benoted that combinations of compounds can give rise to increasedcytotoxicity. AZT and recombinant interferon alpha have an increasedcytotoxic effect on normal human bone marrow progenitor cells.

The compound (2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyridmidin-2-one,also known as 3TC is currently undergoing clinical trials for thetreatment of conditions associated with infection by HIV.

We have now found that 3TC exhibits unexpected advantages when used incombination with certain non-nucleoside inhibitors of HIV.

There is thus provided in a first aspect of the invention a combinationcomprising 3TC or a pharmaceutically acceptable derivitive thereof andnevirapine or a pharmaceutically acceptable derivative thereof.

Nevirapine has the chemical name11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,3-b;2¹,3¹-e][1,4]diazepin-6-one and is also known as BI-RG-587.

3TC will normally be provided substantially free of the corresponding(+)-enantiomer, that is to say no more than about 5% w/w of the(+)-enantiomer, preferably no more than about 2%, in particular lessthan about 1% w/w will be present.

By "a pharmaceutically acceptable derivative" is meant anypharmaceutically acceptable salt, ester, or salt of such ester, of aparent compound or any other compound which, upon administration to therecipient, is capable of providing (directly or indirectly) the parentcompound or an antivirally active metabolite or residue thereof.

It will be appreciated by those skilled in the art that 3TC may bemodified to provide pharmaceutically acceptable derivatives thereof atfunctional groups in both the base moiety and at the hydroxymethyl groupof the oxathiolane ring. Modification at all such functional groups areincluded within the scope of the invention. However of particularinterest are pharmaceutically acceptable derivatives obtained bymodification of the 2-hydroxymethyl group of the oxathiolane ring.

Preferred esters of 3TC include the compounds in which the hydrogen ofthe 2-hydroxymethyl group is replaced by an acyl function R--CO-- inwhich the noncarbonyl moiety R of the ester is selected from hydrogen,straight or branched chain alkyl (e.g. methyl, ethyl, n-propyl, t-butyl,n-butyl), alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl),aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionallysubstituted by halogen, C₁₋₄ alkyl or C₁₋₄ alkoxy); sulphonate esterssuch as alkyl-or aralkylsulphonyl (e.g. methanesulphonyl); amino acidesters (e.g. L-valyl or L-isoleucyl) and mono-, di- or tri-phosphateesters.

With regard to the above described esters, unless otherwise specified,any alkyl moiety present advantageously contains 1 to 16 carbon atoms,particularly 1 to 4 carbon atoms. Any aryl moiety present in such estersadvantageously comprises a phenyl group.

In particular the esters may be a C₁₋₁₆ alkyl ester, an unsubstitutedbenzyl ester or a benzyl ester substituted by at least one halogen(bromine, chlorine, fluorine or iodine), C₁₋₆ alkyl, C₁₋₆ alkoxy, nitroor trifluoromethyl groups.

Pharmaceutically acceptable salts of 3TC include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acids include hydrochloric, hydrobromic, sulphuric,nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic,salicylic, succinic, toluene-p-sulphonic, tartaric, acetic, citric,methanesulphonic, formic, benzoic, malonic, naphthalene-2-sulphonic andbenzenesulphonic acids. Other acids such as oxalic, while not inthemselves pharmaceutically acceptable, may be useful as intermediatesin obtaining the compounds of the invention and their pharmaceuticallyacceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (e.g. sodium),alkaline earth metal (e.g. magnesium), ammonium and NR₄ ⁺ (where R isC₁₋₄ alkyl) salts.

3TC is either synergistic with the second component of the combinationand/or reduces the cytotoxic effects of the second component.

The advantageous effects of 3TC and nevirapine are realised over a wideratio for example 1:250 to 250:1 preferably 1:50 to 50:1, particularlyabout 1:10 to 10:1 by weight. Conveniently each compound will beemployed in the combination in an amount at which it exhibits antiviralactivity when used alone.

It is expected that the present combinations will be generally usefulagainst viral infections or virus-associated tumours in humans, and themethod of their use to inhibit viral infectivity or tumour growth invitro or in vivo is also within the scope of the present invention.

Thus there is provided in a second aspect a method for the treatment ofa viral infection in a mammal, including man, comprisingco-administration of 3TC or a pharmaceutically acceptable derivativethereof and nevirapine or a pharmaceutically acceptable derivativethereof.

It will be appreciated that 3TC and nevirapine may be administeredeither simultaneously (either separately or in combination) orsequentially. If administration is sequential, the delay inadministering the second of the active ingredients should not be such asto lose the benefit of any synergistic effect of the combination.Preferably administration will be simultaneous.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment ofestablished infections or symptoms.

It will be further appreciated that the amount of a combination of theinvention required for use in treatment will vary not only with theparticular compounds selected but also with the route of administration,the nature of the condition being treated and the age and condition ofthe patient and will be ultimately at the discretion of the attendantphysician or veterinarian. In general however a suitable dose will be inthe range of from about 1 to about 750 mg/kg e.g. from about 10 to about75 mg/kg of bodyweight per day, such as 3 to about 120 mg per kilogrambody weight of the recipient per day, preferably in the range of 6 to 90mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day of each ofthe active ingredients of the combination.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example as two,three, four or more sub-doses per day.

The combination is conveniently administered in unit dosage form; forexample containing 10 to 1500 mg, conveniently 20 to 1000 mg, mostconveniently 50 to 700 mg of each active ingredient per unit dosageform.

Ideally the combinations should be administered to achieve peak plasmaconcentrations of each of the active compound of about 1 to about 75 μM,preferably about 2 to 50 μM, most preferably about 3 to about 30 μM.This may be achieved, for example, by the intravenous injection of a 0.1to 5% solution of the active ingredients, optionally in saline, ororally administered as a bolus containing about 1 to about 100 mg ofeach active ingredient. Desirable blood levels may be maintained by acontinuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or byintermittent infusions containing about 0.4 to about 15 mg/kg of eachactive ingredient.

While it is possible that, for use in therapy, the active ingredients ofthe combination may be administered as the raw chemical it is preferableto present combinations as a pharmaceutical formulation.

The invention thus further provides a pharmaceutical formulationcomprising 3TC or a pharmaceutically acceptable derivative thereof andnevirapine or a pharmaceutically acceptable derivative thereof togetherwith one or more pharmaceutically acceptable carriers therefor and,optionally, other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be `acceptable` in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipient thereof.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administrationor in a form suitable for administration by inhalation or insufflation.The formulations may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods include the step of bringinginto association the active compound with liquid carders or finelydivided solid carriers or both and then, if necessary, shaping theproduct into the desired formulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrates, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of, for example, aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The compounds according to the invention may also be formulated forparenteral administration (e.g. by injection, for example bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, prefilled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilising and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

For topical administration to the epidermis the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilising agents, dispersing agents, suspending agents, thickeningagents, or colouring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavoured base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping inmoulds.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active .ingredient such carriers as are known in the artto be appropriate.

For intra-nasal administration the compounds of the invention may beused as a liquid spray or dispersible powder or in the form of drops.

Drops may be formulated with an aqueous or non-aqueous base alsocomprising one more dispersing agents, solubilising agents or suspendingagents. Liquid sprays are conveniently delivered from pressurised packs.

For administration by inhalation the compounds according to theinvention are conveniently delivered from an insufflator, nebuliser or apressurised pack or other convenient means of delivering an aerosolspray. Pressurised packs may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurised aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form in, for example, capsules or cartridges ore.g. gelatin or blister packs from which the powder may be administeredwith the aid of an inhalator or insufflator.

When desired the above described formulations adapted to give sustainedrelease of the active ingredient may be employed.

The pharmaceutical compositions according to the invention may alsocontain other active ingredients such as antimicrobial agents, orpreservatives.

3TC may be obtained as described in International Patent Application No.WO91/17159.

Nevirapine may be obtained as described in Science, 28, 1411-1414.

The following examples illustrate the invention but are not intended asa limitation thereof.

EXAMPLE 1

Antiviral Activities Alone or in Combination

Compounds are first serially-diluted in 2-fold decrements in 96-wellmicrotitre plates. Chequerboard titrations are prepared by mixing 25 μlaliquots from each compound dilution both alone or in combination (to afinal volume of 50 μl in new 96-well microtitre plates). Aliquots ofMT-4 cells (10⁶ cells/ml)in RPMI 1640 growth medium are infected withHIV-1 strain RF at a moi of 2×10⁻³ infectious doses/cell. Virus isadsorbed at room temperature for 90 minutes, after which the cells arewashed in RPMI 1640 growth medium to remove unadsorbed virus andresuspended at 10⁶ cells/ml in RPMI 1640 growth medium. 50 μl ofinfected cell suspension are inoculated into wells containing compoundor growth medium only. 50 μl of mock-infected cell suspension areinoculated into wells not containing compound. The plates are thenincubated for 7 days at 37° C. in 5% CO₂ /air.

After incubation, 10 μl of 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) at 7.5 mg/ml are added to all wellsand the plates incubated for a further 90 minutes at 37° C. 150 μl of10% (v/v) Tdton X-100 in isopropanol are then added and the cellsresuspended. After 15 minutes at room temperature the plates areanalysed in a Multiskan MC (Flow Laboratories, Irvine, UK) reader at 405nm. Conversion of yellow MTT to its formazan derivative is maximum inthe uninfected untreated cells, and absent in untreated infected cells.

Dose-response curves are plotted for each compound alone (IC50% values)and for reciprocal titrations of each compound at a fixed concentrationof the second compound. Isobolograms of all compound combinations givingIC50% values are plotted.

If the, IC50% values of compound combination lies on a line joining theIC50% values of each compound on its own, then the two compounds actadditively. If the combination IC50% lie to the left of the line, thecompounds are acting synergistically.

EXAMPLE 2

Cytotoxicities of Compounds Alone and in Combination.

Cytotoxicity is determined by examination of the cells employed inExample 1 following drug treatment and/or by comparing thecytotoxicities of 3TC and the non-nucleoside HIV inhibitors alone and incombination (at μg/ml ratios of 1:1, 1:5 and 5:1) in uninfectedperipheral blood lymphocytes and an established T-lymphocyte cell line;cytotoxicity is measured using a [³ H]-thymidine uptake assay.

I claim:
 1. A combination comprising about 150 mg(2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt or ester thereof and about 200 mg11-cyclopropyl-5, 11-dihydro-4-methyl-6H-dipyrido[3,3-b;2¹,3¹-e][1,4]diazepin-6-one or a pharmaceutically acceptable salt or esterthereof.
 2. A combination comprising a first compound which is (2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-oneand a second compound which is11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,3-b;2¹,3¹-e][1,4]diazepin-6-one; said combination containing said first andsecond compounds in effective amounts to achieve after oraladministration peak plasma concentrations of from 3 to about 30 μM ofeach of the compounds and to achieve a plasma molar ratio of said firstcompound to said second compound of about 1:1.
 3. A pharmaceuticalformulation comprising a combination as defined in claim 1 together witha pharmaceutically acceptable carrier thereof.
 4. A method for thetreatment of a mammal suffering from or susceptible to infection by HIVcomprising co-administration of about 150 mg(2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt or ester thereof and about 200 mg11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,3-b;2¹ 3¹-e][1,4]diazepin-6-one or a pharmaceutically acceptable salt or esterthereof.
 5. A method according to claim 4, wherein administration issequential.
 6. A method according to claim 4, wherein administration issimultaneous.
 7. A method according to claim 4, wherein the dosages areadministered twice a day.
 8. A method according to claim 4, wherein saidmammal is a man.
 9. A pharmaceutical formulation comprising acombination as defined in claim 2 together with a pharmaceuticallyacceptable carrier therefor.